A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR Abstract: Solid-state NMR (ssNMR) biotechnology is emerging as an alternative method of choice for high- resolution structure determination for integral membrane proteins (IMPs). ssNMR provides a unique platform to investigate protein dynamics, and functional studies of wide range of biomolecules in their supramolecular assemblies. While there exists a suite of magic angle spinning (MAS) and oriented sample (OS) solid state NMR experiments for structural characterization of small- and medium-sized proteins, these methods face several challenges in larger systems. Central to the challenges are NMR sensitivity and resolution. Fast MAS and 1H detected experiments improve sensitivity but are limited by sample volume and relatively poor resolution over small isotropic chemical shift dispersion. Additionally, the efficiency of MAS experiments depends largely on through-bond and through-space coupling constants, solvent suppression, and coherence pathways selection during rotor synchronized multi-pulse applications. They also suffer from sensitivity loss due to local and global motions in proteins. On the other hand, static OS NMR experiments in membrane proteins improve resolution by measuring anisotropic shifts and heteronuclear dipolar couplings, but are limited to dilute spins and low gamma 15N detection only. It has long been realized that unification of MAS and OS has the ability to widen the spectroscopic applications to large globular and membrane proteins. Switched angle spinning (SAS) probes unify MAS, dynamic angle spinning (DAS) and variable angle spinning (VAS) techniques in ssNMR, and potentially correlate isotropic and anisotropic shifts/couplings in more than one Fourier dimension. Such powerful techniques are still far from practical use, because SAS probes in the past have suffered from the lack of reliability due to hardware failures such as the survival of multi-channel rf-leads, rf coil performance including B1 field strength and homogeneity, spinning stability, and lastly rapid reorientation and accurate angle reproducibility. Technical difficulties and engineering challenges thus far have limited the probe technology to only two-frequency channels. This proposal seeks funding for the development of a reliable switched angle spinning probe devoid of previously encountered hardware related issues and compatible with high power pulsed-field gradients. The Phase-I probe will have fixed tuning frequencies for 1H, 13C, and 15N nuclei at 11.7 T for biological applications only. The phase-II probe will advance the technology with two broad-band low-frequency channels to accommodate other functional elements important in biology and chemistry. Additionally, the triple-channel probe will be compatible with standard three-axis gradient coils in order to enable gradient enhanced spectroscopic methods, diffusion NMR, and micro-imaging capabilities in solid state. The advent of such a probe will enhance our ability to develop novel methods for NMR study of proteins and screening of therapeutic drugs.